Microarray technology has become a crucial tool for large-scale and high-throughput biology. A protein-detecting microarray comprises many different affinity reagents (frequently antibodies) arrayed at high spatial density on a solid support. Each agent captures its target protein from a complex mixture (such as serum or cell lysate), and the captured proteins are subsequently detected and quantified. The protein microarray format enables fast, easy and parallel detection of thousands of addressable proteins and side-by-side measurements. It may be applied to analyze antibody-antigen, protein-protein, protein-nucleic acid, protein-lipid and protein-small-molecule interactions, as well as enzyme-substrate interactions.
Currently, the captured proteins in an antibody array are detected in two ways. One is a sandwich immunoassay in which capture antibodies are immobilized on the solid support, and the bound proteins are detected using a second, labeled detection antibody. For example, Huang et al. (Anal. Biochem. 2001, 294:55-62) described an ECL based immunoassay array for the simultaneous assay of 24 cytokines from either cultured media or patient sera. The system was based on the standard sandwich ELISA technology but the initial capture antibodies raised to the various cytokines were transferred in an ordered format onto a membrane. It is evident that this type of antibody array is a simple collection of multiple individual ELISA assays. Another is an antigen capture assay in which proteins are similarly captured by immobilized antibodies, but the captured proteins are detected directly. For example, Haab et al. (Genome Biol., 2001, 2research0004.1-0004.13)labeled two samples independently with distinguishable fluorophores and mixed the samples before applying them to the array. However, this method does not offer signal amplification beyond that provided by the use of fluorophores. In addition, the labeling is time consuming and some proteins may be labeled preferentially on their antigenic epitopes and lose their ability to be captured by their affinity reagents.
Therefore, there is an imperative need to have a system and method of detecting antigens captured on an antibody array, wherein the system and method overcome most, if not all, of the limitations in the prior arts. This invention satisfies this need by disclosing a system and method of detecting antigens captured on an antibody array that are independent of antigen structures. Other advantages of this invention will be apparent with reference to the detailed description.